Age alters dopaminergic responses to estradiol

European Journal of Pharmacology, 82 (1982) 73-75

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Elsevier Biomedical Press

Short communication AGE A L T E R S D O P A M I N E R G I C R E S P O N S E S T O E S T R A D I O L E D W A R D J. ROY *, SUSAN S H E I N K O P and M A R L E N E A. WILSON

Department of Psychology and Neural and Behavioral Biology Program, University of Illinois, Champaign-Urbana, Illinois, U.S.A. Received 2 June 1982, accepted 7 June 1982

E.J. ROY, S. SHEINKOP and M.A. WILSON, Age alters dopaminergic responses to estradiol, European J. Pharmacol. 82 (1982) 73-75. Young (3-5 months) and middle-aged (17-19 months) ovariectomized rats were treated with estradiol benzoate for three days and striatal dopaminergic function was assessed behaviorally (circling in response to apomorphine) and neurochemically (binding of [3H]spiroperidol by striatal membranes). The results suggest that striatal dopaminergic function may respond differently to estrogen in older animals compared to younger animals. Dopamine

Age

Estrogens

1. Introduction

The influence of estrogens on extrapyramidal function is controversial. An estrogen-induced increase in sensitivity of dopaminergic function is suggested by studies of stereotypy or circling behavior activated by dopamine agonists such as amphetamine and apomorphine (Nausieda et al., 1979; Hruska and Silbergeld, 1980). Conversely, estradiol has been reported to cause less response to apomorphine on a measure of circling behavior (Bedard et al., 1978). The work of Gordon (1980) may have clarified the situation by describing a biphasic action of estrogen on stereotypy. Estrogen first suppresses the effects of apomorphine, but after continuous treatment or after the discontinuation of treatment there is an increase in sensitivity to the dopamine agonists. Dopamine receptors are one possible neurochemical site for a change in sensitivity; receptor concentrations have been reported to increase after estrogen treatment (DiPaolo et al., 1981 ; Hruska and Silbergeld, 1980).

Age may be an important consideration in this problem. Dopamine receptors are reduced in aged rodents, either in concentration (Joseph et al., 1978) or in affinity for spiroperidol (Govini et al., 1980). The sensitivity of striatal dopamine systems measured behaviorally might be expected to decline with age, in light of the aging-related disorders of extrapyramidal function such as Parkinson's disease. Joseph et al. (1978) reported a decline with age in circling responses to amphetamine but not to apomorphine, and Kilbey et al. (1980) reported age-related changes in some dopamine stimulated behaviors but not others. The present investigation compared the influence of estradiol on indices of striatal dopaminergic function in young and middle-aged female rats. Apomorphine-induced circling behavior was the behavioral measure, and [3H]spiroperidol was used to assay dopamine receptors in the striatum.

2. Materials and methods * To whom all correspondence should be addressed: Department of Psychology, 603 E. Daniel, University of Illinois, Champaign, Illinois 61820, U.S.A. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

Forty ovariectomized female Long-Evans rats (derived from Simonsen stock) were used. Young rats were four to six months old and weighed

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about 290 g, and old rats were 15-17 months old and weighed about 360g. Long-Evans rats begin to show irregular estrous cycles about 12 months of age, so these rats were clearly middle-aged but not senescent. An apparatus similar to that described by Greenstein and Glick (1975) was used to measure circling behavior. Quarter turns and full turns were recorded as well as the direction of rotation. All animals were injected intraperitoneally with 10 m g / k g of apomorphine, freshly prepared in 0.1% ascorbic acid-saline. Circling behavior was recorded for 30 min, beginning after a 5-min acclimatization period in the arena. Animals were tested in the dark portion of a 14:10 lighting cycle. Estrogen treated animals were injected subcutaneously with 10 t~g/kg estradiol benzoate in sesame oil for three days and tested 24 h after the third injection. They were uninjected for three days and the treatment repeated. Control animals received sesame oil injections. Treatments were reversed the following week. Data were analyzed by analysis of variance to determine effects of age, hormonal treatment, repeated testing, and the order in which hormone treatments were received. Dopamine receptors in the striatum were assayed in 22 randomly selected animals from the behavior experiments using modifications of the method of Fields et al. (1977). After three days of estradiol benzoate (10 /~g/kg) or vehicle treatment, animals were anesthetized and perfused with cold saline. Samples of striatum from individual animals were homogenized with a polytron in a 2 ml Tris-buffered physiological salt solution (50 m M Tris, l m M MgC12, 5 m M KC1, 120 mM NaC1, p H 7.4 at 22°C). Samples were centrifuged at 22000 × g for 20 min, the pellet was washed with 4 m l buffer, and resuspended by brief polytron treatment in 10 ml buffer. Aliquots were incubated with a range of six concentrations of [3H]spiroperidol (New England Nuclear, SA 36 C i / m m o l ) from 0.1-2.0 nM, with parallel tubes containing 100-fold excess unlabelled haloperidol as the competitor to determine specific binding. Samples (700 ~tl, 0.1-0.2 mg p r o t e i n / m l ) were incubated at 37°C, for 30 min, then filtered on W h a t m a n G F / B filters under vacuum, rinsing three times with 2 m l cold buffer. Protein was determined using Coomassie G-250 dye binding.

Binding parameters were determined by least squares curve fitting, with nonspecific binding as a fitted parameter.

3. Results

Analysis of the number of full rotations revealed a significant interaction of age, estrogen treatment, and treatment order (F(1,36)= 6.3275, P < 0.02). That is, although estrogen treatment did not affect the number of rotations during hormonal treatment, there was an increase in rotations after the cessation of hormonal treatment in the young animals but not in the old animals (fig. 1). Estrogen treatment also appeared to affect the dominant direction of rotation. Most animals (24/40) consistently showed a relative preference for one direction through the four tests. Of the 16 animals who switched their dominant direction, 14 switched between two estrogen treatments. Two animals changed dominant direction between

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60

YNG EB - OIL O I L - EB

A--Z~ 0---0

OLD EB - OIL

m--n

OI - - L - E BO - - O / / ~

c/J z O

v<

4O

tO 2O

TRIAL

Fig. 1. Effect of estrogen t r e a t m e n t on a p o m o r p h i n e - i n d u c e d r o t a t i o n s in y o u n g a n d m i d d l e - a g e d female rats (n = 10). Full r o t a t i o n s were recorded for 30 m i n following 10 m g f l k g a p o m o r p h i n e . Two g r o u p s of y o u n g rats and two g r o u p s of old rats were treated w i t h estradiol benzoate a n d oil. One group of each age received estradiol benzoate (EB) for two trials followed by oil for two trials, a n d the second g r o u p of each age received oil followed by EB. There was a significant ( P < 0 . 0 2 ) i n t e r a c t i o n of age, treatment, and t r e a t m e n t order - y o u n g a n i m a l s increased circling following the cessation of h o r m o n e t r e a t m e n t b u t older a n i m a l s did not.

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TABLE 1 Effect of estrogen in young and old rats on binding of [3 H]spiroperidol to striatal dopamine receptors. Animals (n = 5 or 6) were treated as in the behavioral experiments with estradiol benzoate (EB) or oil for three days and then sacrificed. Striatal m e m b r a n e s were incubated with [3 H]spiroperidol (0.1-2.0 nM) with and without 100-fold excess unlabeled haloperidol. Binding curves were analyzed by least squares curve fitting.

Y o u n g oil Y o u n g EB Old oil Old EB

(pM)

Bmax ( f m o l / m g protein)

Ko

687 ~+56 812--63 552±58 573--36

63 -+ 10 68 + 16 52 + 12 107+21

(although our increase did not attain statistical significance) and no change in affinity for spiroperidol. On the other hand, in older animals there was little suggestion of an effect of estrogen on dopamine receptor concentration. However, estrogen treatment caused a reduction in the affinity of dopamine receptors for spiroperidol in older animals. Taken together the behavioral and biochemical data suggest an altered responsiveness of the striatal dopaminergic system to estrogen in middle-aged animals.

Acknowledgements estrogen and oil treatments; none switched between oil treatments. The [3 H]spiroperidol binding data (table 1) were best fit by a one-site model. There was an overall reduction in the concentration of striatal dopamine receptors in older animals (P